Abstract. The cell adhesion receptor integrin ?V?8 is important for vascular and cardiac morphogenesis, lung epithelial homeostasis, CNS development, immune function, and latent transforming growth factor beta (TGF-?) activation. Ligand binding and integrin activation are coupled in most integrins to two distinct, large conformational changes: extension at the bent integrin knees and opening of the ligand-binding headpiece. Thus, most integrins can equilibrate between two low affinity conformations, bent-closed and extended-closed, and a high affinity extended-open conformation. However, integrin ?V?8 is atypical because it is constitutively extended and resistant to ligand-induced headpiece opening. Moreover, the ?8 cytoplasmic domain lacks binding motifs for the cytoskeletal adaptors talin and kindlin, but binds a distinct cytoskeletal protein associated with lung adenocarcinoma, DAL-1. We will structurally and functionally characterize ?V?8 to determine the molecular mechanisms that govern its atypical behavior. In Aim 1, we obtain an overview of ?V?8 ectodomain and headpiece conformational states compared to ?V?6 by EM. We investigate whether binding of pro-TGF-?1 or a crossreacting, de novo designed ?V?6-binding protein (?V?6-BP) induces headpiece opening of ?V?8. We mutationally examine whether divergent sequences in ?8 metal-binding sites or in the ?I-hybrid interface stabilize the closed headpiece and whether divergent sequence at the ?-knee stabilizes the extended conformation. In Aim 2, we obtain crystal structures of the ?V?8 headpiece, its complexes with peptide ligand, pro-TGF-?1, or ?V?6-BP and a structure of the ?V?8 ectodomain. Structures will demonstrate how ?V?8 resists ligand-induced headpiece opening and maintains constitutive extension. In Aim 3, we investigate affinity and functional regulation of ?V?8 and transition to developing a potential therapeutic. We characterize the structure of the ?8 cytoplasmic domain bound to DAL-1 and investigate the role of this interaction in ?V?8-mediated activation of latent pro-TGF-?1. We hypothesize that in vivo, the ?V?8 headpiece may be opened by inside-out signaling, and increase affinity for ligand. To establish whether headpiece opening of ?V?8 increases affinity, we will use mutations to release restraints on ?V?8 headpiece opening, measure their effect on affinity for ligand, and correlate increases in affinity with headpiece opening measured by EM. Structures of ?V?8 bound to current, highly potent ?V?6 and ?V?8 small molecule antagonists will explain their limited selectivity for ?V?6 and ?V?8. The insights obtained will guide development of second generation, highly selective, pure antagonist therapeutics to treat diseases such as chronic obstructive pulmonary disease.